Biomedical subjects

Qunfang Li

Publications and source records attributed to Qunfang Li.

6 recordsLinked to original sources

PubMed2006

Stat1 expression is not sufficient to regulate the interferon signaling pathway in cellular immortalization.

DNA hypermethylation in gene promoters is an epigenetic mechanism regulating gene expression in cellular immortalization, an important step in carcinogenesis. Previously, we studied the genes dysregulated during immortalization using spontaneously immortalized fibroblasts from patients with Li-Fraumeni syndrome (LFS), who carry a germline mutation in the tumor suppressor gene p53. We found that multiple interferon (IFN) signaling pathway genes were regulated by epigenetic silencing. In this study we focused on a key regulator of that pathway, the signal transducer and transcription activator 1 (Stat1) gene. Although Stat1 is downregulated after cellular immortalization and upregulated in immortal MDAH041 cells after 5-aza-2'-deoxycytidine (5-aza-dC) treatment, we detected no methylation of the Stat1 promoter region in these cells before or after immortalization. To analyze the function of Stat1 in immortalization, we expressed Stat1 in immortal MDAH041 cells by stable infection, expecting to induce IFN-regulated genes or cellular senescence or both. However, the overexpression of Stat1 alone was not sufficient to repress the proliferation rate of immortal MDAH041 cells or induce senescence in immortal MDAH041 cells. We concluded that factor(s) additional to Stat1 (whether IFN dependent or not) are required for the immortalization of LFS fibroblasts.

Animals↗

PubMed2006

Expression profiling identifies three pathways altered in cellular immortalization: interferon, cell cycle, and cytoskeleton.

Abrogation of cellular senescence, resulting in immortalization, is a necessary step in the tumorigenic transformation of a cell. Four independent, spontaneously immortalized Li-Fraumeni syndrome (LFS) cell lines were used to analyze the gene expression changes that may have given these cell lines the growth advantage required to become immortal. A cellular senescence-like phenotype can be induced in immortal LFS cells by treating them with the DNA methyltransferase (DNMT) inhibitor 5-aza-deoxycytidine. We hypothesized, therefore, that genes epigenetically silenced by promoter methylation are potentially key regulators of senescence. We used microarrays to compare the epigenetic gene expression profiles of precrisis LFS cells with immortal LFS cells. Gene ontology analysis of the expression data revealed a statistically significant contribution of interferon pathway, cell cycle, and cytoskeletal genes in the process of immortalization. The identification of the genes and pathways regulating immortalization will lead to a better understanding of cellular immortalization and molecular targets in cancer and aging.

Azacitidine↗

PubMed2004

Development of an autocrine neuregulin signaling loop with malignant transformation of human breast epithelial cells.

Neuregulin (NRG) is a heparin-binding factor that activates members of the epidermal growth factor family of tyrosine kinase receptors including erbB2 that is overexpressed in more aggressive types of breast cancer. The exact role that NRG plays in breast cancer is complicated by the fact that NRG has been shown to have both proliferative and antiproliferative effects, depending on the breast cancer cell line used. Using an isogenic series of breast epithelial cell lines (MCF10A) ranging from benign to malignant, we found that the actions of NRG changed from antiproliferative to proliferative as the cells progress to cancer. This correlated with a progressive inability of NRG to down-regulate a group of proliferation genes identified previously using cDNA microarrays. As the cells progress to malignancy, they expressed higher levels of erbB2 and lower levels of erbB3 and secreted high levels of NRG into the culture media, resulting in high basal levels of erbB receptor phosphorylation. Disruption of this autocrine signaling loop by blocking ligand-induced receptor activation inhibited cancer cell proliferation. These results demonstrate that the transition of MCF10A cells from normal to premalignant to malignant correlates with the development of a constitutively active autocrine NRG signaling loop that promotes cell proliferation and suggest that disrupting this autocrine loop may provide an important therapeutic measure to control breast cancer cell growth.

Breast↗

PubMed2004

Accuracy of cDNA microarray methods to detect small gene expression changes induced by neuregulin on breast epithelial cells.

BACKGROUND: cDNA microarrays are a powerful means to screen for biologically relevant gene expression changes, but are often limited by their ability to detect small changes accurately due to "noise" from random and systematic errors. While experimental designs and statistical analysis methods have been proposed to reduce these errors, few studies have tested their accuracy and ability to identify small, but biologically important, changes. Here, we have compared two cDNA microarray experimental design methods with northern blot confirmation to reveal changes in gene expression that could contribute to the early antiproliferative effects of neuregulin on MCF10AT human breast epithelial cells. RESULTS: We performed parallel experiments on identical samples using a dye-swap design with ANOVA and an experimental design that excludes systematic biases by "correcting" experimental/control hybridization ratios with control/control hybridizations on a spot-by-spot basis. We refer to this approach as the "control correction method" (CCM). Using replicate arrays, we identified a decrease in proliferation genes and an increase in differentiation genes. Using an arbitrary cut-off of 1.7-fold and p values <0.05, we identified a total of 32 differentially expressed genes, 9 with the dye-swap method, 18 with the CCM, and 5 genes with both methods. 23 of these 32 genes were subsequently verified by northern blotting. Most of these were <2-fold changes. While the dye-swap method (using either ANOVA or Bayesian analysis) detected a smaller number of genes (14-16) compared to the CCM (46), it was more accurate (89-92% vs. 75%). Compared to the northern blot results, for most genes, the microarray results underestimated the fold change, implicating the importance of detecting these small changes. CONCLUSIONS: We validated two experimental design paradigms for cDNA microarray experiments capable of detecting small (<2-fold) changes in gene expression with excellent fidelity that revealed potentially important genes associated with the anti-proliferative effects of neuregulin on MCF10AT breast epithelial cells.

Blotting, Northern↗

PubMed2004

Synergistic effects of neuregulin and agrin on muscle acetylcholine receptor expression.

The proper function of neuromuscular junctions requires an extremely high density of acetylcholine receptors (AChRs) that may be achieved from neuron-derived factors including agrin and neuregulin. Here, we show that neuregulin-1 and agrin co-localize at neuromuscular junctions in vivo and form complexes when co-transfected into COS-7 cells. When these COS-7 cells are cultured with myotubes, synergistic effects are observed for AChR clustering, membrane insertion of new AChRs, and induction of AChR mRNA. Even a muscle form of agrin that lacks intrinsic clustering activities by itself, significantly enhances neuregulin-induced clustering and insertion of AChRs. While the heparin-binding (A) domain of agrin is required for agrin localization in the extracellular matrix adjacent to AChR clusters, the heparan sulfate-containing domain of agrin is needed for the synergistic effects and co-localization with neuregulin-1. These results suggest that matrix interactions between exogenously supplied agrin and neuregulin-1 on the muscle surface provide a localized source of signaling factors needed to produce high densities of AChRs at neuromuscular junctions.

Agrin↗

PubMed2004

Highly selective thiocyanate electrode based on bis-[N-(2-hydroxyethyl)salicylaldimino]copper(II) complex as a neutral carrier.

A novel selective thiocyanate electrode of a PVC membrane based on bis-[N-(2-hydroxyethyl)salicylaldimino]copper(II) [Cu(II)-BNSDM] as a neutral carrier is reported. The selectivity sequence of this electrode is as follows: SCN- > ClO4- > I- > Sal- > Br- > NO2- > Cl- > NO3- > H2PO4- > SO4(2-), which is an anti-Hofmeister selectivity sequence. The electrode exhibits a Nernstian potential linear range to thiocyanate from 1.0 x 10(-1) to 6.0 x 10(-6) mol/L with a detection limit of 2.0 x 10(-6) mol/L and a slope of (-59.0+/-0.2)mV/decade in pH 5.0 of a phosphate buffer solution at 25 degrees C. Electrochemical impedance spectroscopy (EIS) and UV spectroscopy techniques were performed to understand the response mechanism of the electrode. The electrode had the advantage of simplicity, high selectivity, fast response and low detection limit compared with the other electrode, which had been reported before. The electrode had been applied to wastewater analysis, and the obtained results were satisfactory.

Journal Article↗